Rational Design and Multicomponent Synthesis of Lipid-Peptoid Nanocomposites towards a Customized Drug Delivery System Assembly.

Nanotechnology has assumed a significant role over the last decade in the development of various technologies applied to health sciences. This becomes even more evident with its application in controlled drug delivery systems. In this context, peptoids are a promising class of compounds for application as nanocarriers in drug delivery systems. These compounds can be obtained efficiently and with highly functionalized structural diversity via the Ugi 4-component reaction (U-4CR). Herein, we report the design of the process control strategy for the future development of lipid-peptoid-based customized drug delivery system assemblies. Over 20 lipid-peptoid nanocomposites were synthesized via the U-4CR in good to excellent yields. These products were successfully submitted to the nanoparticle formation by the emulsification-evaporation process from lipophilic solution and analyzed via Dynamic Light Scattering (DLS). Several molecules generated nanoparticles with a size ≤200 nm, making them good candidates for drug delivery systems, such as in cancer treatment.

Detection of carbapenemase- and ESBL-producing Klebsiella pneumoniae from bovine bulk milk and comparison with clinical human isolates in Italy.

Klebsiella pneumoniae is the most common Klebsiella species infecting animals and is one of the causing agents of mastitis in cows. The rise of antimicrobial resistance in K. pneumoniae, particularly in strains producing extended-spectrum ß-lactamases (ESBLs) and/or carbapenemases, is of concern worldwide. Recently (Regulation UE No 2022/1255), carbapenems and cephalosporins in combination with ß-lactamase inhibitors have been reserved only to human treatments in the European Union. The aim of this study was to investigate the role of cattle as carrier of human pathogenic carbapenem-resistant (CR) and ESBL-producing K. pneumoniae. On this purpose, a study involving 150 dairy farms in Parma province (Northern Italy) and 14 non replicate K. pneumoniae isolates from patients admitted at Parma University-Hospital was planned. Four multidrug resistant (MDR) K. pneumoniae strains were detected from 258 milk filters collected between 2019 and 2021. One carbapenemase KPC-3-positive K. pneumoniae ST307 (0.4 %; 95 % CI - 0.07 - 2.2) was detected in milk filters. The isolate also harboured OXA-9, CTX-M-15 and SHV-106 determinants, together with genes conferring resistance to aminoglycosides (aac(3')-IIa, aph (3″)-Ib, aph (6)-Id), fluoroquinolones (oqxA, oqxB, qnrB1), phosphonic acids (fosA6), sulphonamides (sul2), tetracyclines (tet(A)6) and trimethoprim (dfrA14). One KPC-3-producing K. pneumoniae ST307 was identified also among the human isolates, thus suggesting a possible circulation of pathogens out of the clinical settings. The remaining three bovine isolates were MDR ESBL-producing K. pneumoniae characterized by different genomic profiles: CTX-M-15, TEM-1B and SHV-187 genes (ST513); CTX-M-15 and SHV-145 (ST307); SHV-187 and DHA-1 (ST307). Occurrence of ESBL-producing K. pneumoniae in milk filters was 1.2 % (95 % CI 0.4-3.4). All the isolates showed resistance to aminoglycosides, 3rd-generation cephalosporins, and fluoroquinolones. Among the human isolates, two multidrug resistant ESBL-producing K. pneumoniae ST307 were found, thus confirming the circulation of this high-risk lineage between humans and cattle. Our findings suggest that food-producing animals can carry human pathogenic microorganisms harboring resistance genes against carbapenems and 3rd-generation cephalosporins, even if not treated with such antimicrobials. Moreover, on the MDR K. pneumoniae farms, the antimicrobial use was much higher than the Italian median value, thus highlighting the importance of a more prudent use of antibiotics in animal productions.

Application of different DNA extraction procedures, library preparation protocols and sequencing platforms: impact on sequencing results.

In this study three DNA extraction procedures, two library preparation protocols and two sequencing platforms were applied to analyse six bacterial cultures and their corresponding DNA obtained as part of a proficiency test. The impact of each variable on sequencing results was assessed using the following parameters: reads quality, assembly and alignment statistics; number of single nucleotide polymorphisms (SNPs), detected applying assembly- and alignment-based strategies; antimicrobial resistance genes (ARGs), identified on de novo assemblies of all sequenced genomes. The investigated nucleic acid extraction procedures, library preparation kits and sequencing platforms do not significantly affect de novo assembly statistics and number of SNPs and ARGs. The only exception was observed for two duplicates, which were associated to one PCR-based library preparation kit. Results from this comparative study can support researchers in the choice toward the available pre-sequencing and sequencing options, and might suggest further comparisons to be performed.

Bone marrow failure may be caused by chromosome anomalies exerting effects on RUNX1T1 gene.

BACKGROUND: The majority of the cases of bone marrow failure syndromes/aplastic anaemias (BMFS/AA) are non-hereditary and considered idiopathic (80-85%). The peripheral blood picture is variable, with anaemia, neutropenia and/or thrombocytopenia, and the patients with idiopathic BMFS/AA may have a risk of transformation into a myelodysplastic syndrome (MDS) and/or an acute myeloid leukaemia (AML), as ascertained for all inherited BMFS. We already reported four patients with different forms of BMFS/AA with chromosome anomalies as primary etiologic event: the chromosome changes exerted an effect on specific genes, namely RUNX1, MPL, and FLI1, leading to the disease. RESULTS: We report two further patients with non-hereditary BM failure, with diagnosis of severe aplastic anaemia and pancytopenia caused by two different constitutional structural anomalies involving chromosome 8, and possibly leading to the disorder due to effects on the RUNX1T1 gene, which was hypo-expressed and hyper-expressed, respectively, in the two patients. The chromosome change was unbalanced in one patient, and balanced in the other one. CONCLUSIONS: We analyzed the sequence of events in the pathogenesis of the disease in the two patients, including a number of non-haematological signs present in the one with the unbalanced anomaly. We demonstrated that in these two patients the primary event causing BMFS/AA was the constitutional chromosome anomaly. If we take into account the cohort of 219 patients with a similar diagnosis in whom we made cytogenetic studies in the years 2003-2017, we conclude that cytogenetic investigations were instrumental to reach a diagnosis in 52 of them. We postulate that a chromosome change is the primary cause of BMFS/AA in a not negligible proportion of cases, as it was ascertained in 6 of these patients.

[Eyelid retraction of neurologic origin: Report of three cases]. / Retracción palpebral de causa neurológica: Presentación de tres pacientes, interpretación patogénica.

Eyelid retraction, has received limited attention and it has passively been interpreted as the result of an overactive levator palpebrae superioris muscle secondary to midbrain injury. However, eyelid retractions can occur in other neurological diseases, not directly related with the midbrain. We report three patients who developed eyelid retraction. One patient had a bilateral eyelid retraction, related with Creutzfeldt-Jakob disease (CJD). Another patient had a unilateral right eyelid retraction associated with a thalamic-mesencephalic infarct. The third patient had a bilateral pontine infarction on magnetic resonance imaging. In the patient with CJD, eyelid retraction did not subside. Among patients with infarctions, the retraction persisted after focal symptoms had subsided, showing an evolution that was apparently independent of the basic process. The analysis of these patients allows us to conclude that the pathogenesis of eyelid retraction includes supranuclear mechanisms in both the development and maintenance of the phenomenon. Unilateral or bilateral eyelid retraction does not alter the normal function of eyelid, which ever had normal close eye blink. In these reported cases, a hyperactivity of levator palpebrae superioris muscle was clinically ruled out.

Retracción palpebral de causa neurológica: presentación de tres pacientes, interpretación patogénica / Eyelid retraction of neurologic origin: report of three cases

Eyelid retraction, has received limited attention and it has passively been interpreted as the result of an overactive levator palpebrae superioris muscle secondary to midbrain injury. However, eyelid retractions can occur in other neurological diseases, not directly related with the midbrain. We report three patients who developed eyelid retraction. One patient had a bilateral eyelid retraction, related with Creutzfeldt-Jakob disease (CJD). Another patient had a unilateral right eyelid retraction associated with a thalamic-mesencephalic infarct. The third patient had a bilateral pontine infarction on magnetic resonance imaging. In the patient with CJD, eyelid retraction did not subside. Among patients with infarctions, the retraction persisted after focal symptoms had subsided, showing an evolution that was apparently independent of the basic process. The analysis of these patients allows us to conclude that the pathogenesis of eyelid retraction includes supranuclear mechanisms in both the development and maintenance of the phenomenon. Unilateral or bilateral eyelid retraction does not alter the normal function of eyelid, which ever had normal close eye blink. In these reported cases, a hyperactivity of levator palpebrae superioris muscle was clinically ruled out.

A genome-wide nanotoxicology screen of Saccharomyces cerevisiae mutants reveals the basis for cadmium sulphide quantum dot tolerance and sensitivity.

The use of cadmium sulphide quantum dots (CdS QDs) is increasing, particularly in the electronics industry. Their size (1-10 nm in diameter) is, however, such that they can be taken up by living cells. Here, a bakers' yeast (Saccharomyces cerevisiae) deletion mutant collection has been exploited to provide a high-throughput means of revealing the genetic basis for tolerance/susceptibility to CdS QD exposure. The deletion of 112 genes, some associated with the abiotic stress response, some with various metabolic processes, some with mitochondrial organization, some with transport and some with DNA repair, reduced the level of tolerance to CdS QDs. A gene ontology analysis highlighted the role of oxidative stress in determining the cellular response. The transformation of sensitive mutants with centromeric plasmids harbouring DNA from a wild type strain restored the wild type growth phenotype when the complemented genes encoded either HSC82, DSK2 or ALD3. The use of these simple eukaryote knock-out mutants for functional toxicogenomic analysis will inform studies focusing on higher organisms.

Modified-atmosphere packaging of hen table eggs: effects on pathogen and spoilage bacteria.

As part of a more comprehensive research activity on the use of modified-atmosphere packaging for the improvement of quality and functional properties of table eggs, the effects of air, 100% CO(2), and 100% O(2) packaging were also evaluated on the survival of experimentally inoculated pathogen bacteria (Salmonella Enteritidis, Escherichia coli, and Listeria monocytogenes) as well as on spoilage bacteria (total aerobic mesophilic bacteria) on table eggs during 30 d of storage at 4, 25, and 37°C by colony count method. In general, temperatures played a major role, rather than gasses, in influencing the bacterial survival. In particular, the lowest microbial loads were registered at 4°C on E. coli and spoilage bacteria, whereas 37°C was the best storage temperature to avoid the psychrotropic microorganism L. monocytogenes development regardless of the gas used. One hundred percent CO(2) packaging, in association with a low storage temperature (4°C), had a significant positive effect in reducing Salmonella loads. On eggs inoculated with L. monocytogenes and stored at 4°C as well as on eggs containing only spoilage bacteria and stored at 25°C, 100% CO(2) resulted the best gas in comparison with air and O(2). One hundred percent CO(2) packaging showed no negative effect on pathogen survival compared with air. Although further improvements are required to control RH within packaging to limit bacteria growth/survival, in view of the positive effects of CO(2) packaging on quality traits of table eggs, 100% CO(2) packaging might represent a promising innovative technique for the maintenance of egg characteristics during transport, retail, and domestic storage.

OTX1 expression in breast cancer is regulated by p53.

The p53 transcription factor has a critical role in cell stress response and in tumor suppression. Wild-type p53 protein is a growth modulator and its inactivation is a critical event in malignant transformation. It has been recently demonstrated that wild-type p53 has developmental and differentiation functions. Indeed an over-expression of p53 in tumor cells induces asymmetrical division avoiding self-renewal of cancer stem cells (CSCs) and instead promoting their differentiation. In this study, 28 human breast carcinomas have been analyzed for expression of wild-type p53 and of a pool of non-clustered homeobox genes. We demonstrated that orthodenticle homolog 1 gene (OTX1) is transcribed in breast cancer. We established that the p53 protein directly induces OTX1 expression by acting on its promoter. OTX1 has been described as a critical molecule for axon refinement in the developing cerebral cortex of mice, and its activity in breast cancer suggests a synergistic function with p53 in CSC differentiation. Wild-type p53 may regulate cellular differentiation by an alternative pathway controlling OTX1 signaling only in breast cancer cells and not in physiological conditions.

Hot air treatment for surface decontamination of table eggs experimentally infected with Salmonella, Listeria, and Escherichia coli.

Hot-air pasteurization was investigated in the EU-funded project "Reducing Egg Susceptibility to Contaminations in Avian Production in Europe (RESCAPE)" as an innovative treatment for surface bacterial decontamination of table eggs. Possible side effects of the treatment on egg quality traits were also studied. The decontamination power of hot air was evaluated over 1 month on shell eggs that were experimentally inoculated with Salmonella enteritidis, Escherichia coli, or Listeria monocytogenes. The S. Enteritidis and L. monocytogenes populations on the surfaces of treated eggs showed a significant reduction compared with untreated eggs. No statistically significant results were obtained comparing E. coli loads on treated and untreated eggs. No detrimental effects on quality traits either immediately after treatment or after 28 days of storage at 20 degrees C were recorded.

The isochromosome i(7)(q10) carrying c.258+2t>c mutation of the SBDS gene does not promote development of myeloid malignancies in patients with Shwachman syndrome.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder, characterized by exocrine pancreatic insufficiency, skeletal abnormalities and bone marrow (BM) dysfunction with an increased risk to develop myelodysplastic syndrome and/or acute myeloid leukaemia (MDS/AML). SDS is caused, in nearly 90% of cases, by two common mutations (that is, c.183_184TA>CT and c.258+2T>C) in exon 2 of the SBDS gene, localized on chromosome 7. Clonal chromosome anomalies are often found in the BM of SDS patients; the most frequent is an isochromosome for long arms of chromosome 7, i(7)(q10). We studied eight patients with SDS carrying the i(7)(q10) who were compound heterozygotes for SBDS mutations. By assessing the parental origin of the i(7)(q10) using microsatellite analysis, we inferred from the results which mutation was present in double dose in the isochromosome. We demonstrate that in all cases the i(7)(q10) carries a double dose of the c.258+2T>C, and we suggest that, as the c.258+2T>C mutation still allows the production of some amount of normal protein, this may contribute to the low incidence of MDS/AML in this subset of SDS patients.

Phage types, ribotypes and tetracycline resistance genes of Salmonella enterica subsp. enterica serovar Typhimurium strains isolated from different origins in Italy.

The tetracycline resistance (tet) gene patterns of 52 tetracycline resistant Salmonella enterica subsp. enterica (S.) serovar Typhimurium isolates collected from animals, food of animal origin, and humans in Italy, were investigated to evaluate whether the tet gene patterns could be used for strain differentiation in addition to phage typing and ribotyping. The detection of tet genes was performed by specific PCR assays. Ribotyping was performed automatically using PvuII as restriction enzyme. Ten different ribotyping patterns were detected. All isolates were positive for at least one of the tet genes studied and six different tet gene patterns were observed. Ribotyping and tet gene patterns showed discriminatory indices of 0.741 and 0.812, respectively. Multiple tet genes were commonly found among tetracycline resistant S. typhimurium isolates from various sources. The resulting tet gene patterns allowed further discrimination of strains which were otherwise indistinguishable by their phage type, ribotype and origin. Thus, the analysis of tet gene patterns might represent an additional tool for the differentiation of S. typhimurium isolates.

Meiotic origin of trisomy in neoplasms: evidence in a case of erythroleukaemia.

Trisomic cells in neoplasms may represent abnormal clones originated from a tissue-confined mosaicism, and arise therefore by a meiotic error. We report on a 16-month-old child with erythroleukaemia (AML-M6), whose marrow karyotype at onset was 48,XX,del(13)(q12q14),del(14)(q22q32),+21,+21. The parental origin of the supernumerary chromosomes 21 was investigated by comparing 10 polymorphic loci scattered along the whole chromosome on the patient's marrow and her parents' leukocytes. Three loci were informative for the presence of three alleles, two of which were of maternal origin; two further loci showed a maternal allele of higher intensity. Lymphocytes and skin fibroblasts showed a normal karyotype, and molecular analysis on leukocytes at remission, buccal smear and urinary sediment cells consistently showed only one maternal allele, whereas neonatal blood from Guthrie spot showed two maternal alleles as in the marrow. An accurate clinical re-evaluation confirmed a normal phenotype. Our results indicate that tetrasomy 21 arose from a marrow clone with trisomy 21 of meiotic origin. To the best of our knowledge, this is the first evidence that supernumerary chromosomes in neoplastic clones may in fact be present due to a meiotic error. This demonstrates that a tissue-confined constitutional mosaicism for a trisomy may indeed represent the first event in multistep carcinogenesis.

Familial partial monosomy 7 and myelodysplasia: different parental origin of the monosomy 7 suggests action of a mutator gene.

Two sisters are reported, both with a myelodysplastic syndrome (MDS) associated with partial monosomy 7. A trisomy 8 was also present in one of them, who later developed an acute myeloid leukemia (AML) of the M0 FAB-type and died, whereas the other died with no evolution into AML. Besides FISH studies, microsatellite analysis was performed on both sisters to gather information on the parental origin of the chromosome 7 involved in partial monosomy and of the extra chromosome 8. The chromosomes 7 involved were of different parental origin in the two sisters, thus confirming that familial monosomy 7 is not explained by a germ-line mutation of a putative tumor-suppressor gene. Similar results were obtained in two other families out of the 12 reported in the literature. Noteworthy is the association with a mendelian disease in 3 out of 12 monosomy 7 families, which suggest that a mutator gene, capable of inducing both karyotype instability and a mendelian disorder, might act to induce chromosome 7 anomalies in the marrow. We postulate that, in fact, an inherited mutation in any of a group of mutator genes causes familial monosomy 7 also in the absence of a recognized mendelian disease, and that marrow chromosome 7 anomalies, in turn, lead to MDS/AML.

17q21-qter trisomy is an indicator of poor prognosis in acute myelogenous leukemia.

A reciprocal translocation (9;11) is often found in acute myeloid leukemia (AML), mostly of the M5a type. We report a case of a child with AML, in whom t(9;11) was observed at diagnosis as the sole structural abnormality, together with trisomies 19 and 21. The diagnosis was AML evolving from a myelodysplastic syndrome (MDS), and the blast morphology was undifferentiated. Chemotherapy failed to induce morphological remission and the patient's condition soon worsened. A subclone appeared and expanded during the course of the disease, with an additional unbalanced translocation (1;17) leading to trisomy of the long arm of chromosome 17 (17q). The data available from the literature on acquired anomalies involving 17q and our observation led us to postulate a specific link between the gain of 17q and complete chemoresistance.

Isochromosome (7)(q10) in Shwachman syndrome without MDS/AML and role of chromosome 7 anomalies in myeloproliferative disorders.

Shwachman syndrome (SS) is an autosomal recessive disorder in which bone marrow dysfunction is observed, with development of myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML) in up to one third of the cases. Inconclusive data are available as to increased chromosome breakage in SS, while chromosome 7 anomalies, and often an isochromosome (7)(q10), are frequent in cases with MDS/AML. We report on the consistent presence of an i(7)(q10) in the bone marrow and blood lymphocytes in one of two sisters affected with SS without any clinical or cytological signs of MDS/AML. Thus, this patient was either a case of constitutional mosaicism for the i(7)(q10), or this had to be acquired in a nondysplastic and non-neoplastic marrow clone. DNA polymorphism analysis demonstrated the paternal origin of the i(7q). We postulate that the SS mutation acts as a mutator gene, and causes karyotype instability; abnormal clones would thus arise in the marrow, and chromosome 7 anomalies, i(7q) in particular, will in turn lead to MDS/AML. If this interpretation is correct, it would be also an indication to consider chromosome 7 anomalies in general, out of SS, as primary changes in MDS/AML pathogenesis.

Full cytogenetic characterization of a new neuroblastoma cell line with a complex 17q translocation.

Recent studies have shown that structural abnormalities of chromosome 17 resulting in gain of material are the most frequent genetic changes in neuroblastoma. We have established a new neuroblastoma cell line from a patient whose disease had evolved from stage 4s to 4, without evidence of deletion of the short arm of chromosome 1 and MYCN amplification, which are considered the most typical genetic indicators of aggressive disease. The cytogenetic study allowed a full characterization of the chromosome changes, and revealed a complex translocation of chromosome 17 leading to a derivative marker which may be described as follows: der(11)t(11;17)(p15;q12)t(11;17) (q22;q12). This resulted in a gain of part of the long arms of chromosome 17, which was recently associated with poor prognosis.